2005 Fiscal Year Final Research Report Summary
New strategy for oxidative stress studies with a newly developed device for O_2^- generation
| Project/Area Number |
15300164
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biomedical engineering/Biological material science
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| Research Institution | Ehime University |
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Principal Investigator |
TAMURA Minoru Ehime University, Sch.of Sci.& Engineer., Assoc.Prof., 理工学研究科, 助教授 (00128349)
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| Co-Investigator(Kenkyū-buntansha) |
MIURA Satoshi Yokohama City Univ., Fac.Medicine, Assoc.Prof., 医学部, 準教授 (60157427)
NAKAMURA Yoichi Osaka Pref.Univ., Life & Environmental Science, Prof., 生命環境科学部, 教授 (90180413)
ROKUTAN Kazuhito Tokushima Univ., Sch. of Health & Bio science, Prof., 大学院・ヘルスバイオサイエンス研究部, 教授 (10230898)
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| Project Period (FY) |
2003 – 2005
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| Keywords | Superoxide / Reactive oxygen intermediate / NADPH oxidase / Cytochrome b_<558> / Fusion protein / Oxidative stress / Cell culture / Molecular device |
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| Research Abstract |
Application of the device to cell experiments
We examined the effect of reactive oxygen species on several cultured cells using the original device. When it was applied to HEK293 cells, apparent cell death took place after 30h. When it was used for CHO cells, cell growth was suppressed but cell death did not occur at the same concentration. On the other hand, the device did not affect growth or viability of Hela cells. These results suggested that the effect of reactive oxygen species varies depending on cell types and carcinoma cells were fairly resistant to reactive oxygen species. Using SOD or catalase in the experiments with HEK293 cells we found that reactive oxygen species that is actually effective is hydrogen peroxide.
Development of device II, the second version of the original device
Unfortunately, the original device was found to be unstable in cell culture media. Therefore, we tried to improve the device in stability by chemical cross-linking. The device, somewhat diluted, was treated with a cross-linker EDC and sulfo-NHS. The resulting device became very stable in a culture medium (e.g.MEM). The half-life of the activity was over 3h and the device showed 40% activity even after 5h. The new device was referred to as "device II".
Application of device II to oxidative stress experiments
The new device was applied to human HL-60 cells and examined the effect of reactive oxygen on the cells. After incubation with device II and NADPH at 22h, cell death was observed and the cell number decreased to 30% of the initial. When the device was added without NADPH, such phenomena was not seen. SOD did not influence the effect much, but catalase blocked the effect mostly, indicating that the actual reacting species is also hydrogen peroxide.
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